Cross-talk between the Platelet-derived Growth Factor and the Insulin Signaling Pathways in 3T3-L1 Adipocytes

Abstract

Phosphatidylinositol (PI) 3-kinase is activated by various growth factors such as PDGF (platelet-derived growth factor) and insulin. The aim of the present study was to determine whether PDGF could modulate insulin activation of PI 3-kinase in 3T3-L1 adipocytes. When cells were preincubated for 5-15 min with PDGF, PI 3-kinase activity associated to insulin receptor substrate 1 (IRS 1) in response to insulin was decreased, due to reduced association of the PI 3-kinase p85 subunit with IRS 1. In addition, following this PDGF pretreatment, the tyrosine phosphorylation of IRS 1 in response to insulin and its electrophoretic mobility were diminished. The change in the mobility of IRS 1 could be attributed to PDGF-induced serine/threonine phosphorylation of the protein which was partly inhibited by PI 3-kinase inhibitors. By contrast, epidermal growth factor, which does not stimulate PI 3-kinase, had no effect on the association of PI 3-kinase with IRS 1 in response to insulin. This series of results indicates that the PDGFinduced serine/threonine phosphorylation of IRS 1 could be due to activation of PI 3-kinase pathway. Furthermore, this phosphorylation of IRS 1 is associated with a decrease in its tyrosine phosphorylation by insulin and in its association with the p85 subunit of PI 3-kinase. This study suggests that a cross-talk exists between the different pathways stimulated by PDGF and insulin in intact cells. Phosphatidylinositol (PI) 3-kinase1 is a common element of the signaling pathway of a large number of tyrosine kinase receptors. PI 3-kinase is a heterodimer consisting of an 85-kDa regulatory subunit (p85) containing one Src homology 3 (SH3) domain and two Src homology 2 (SH2) domains (1-3) and an 110-kDa catalytic subunit (4). The catalytic subunit phosphorylates inositol lipids at the D-3 position of the inositol ring and has been shown to possess a serine kinase activity (5). By contrast, the p85 regulatory subunit functions as an adaptor which, via its SH2 domains, links PI 3-kinase to tyrosinephosphorylated proteins such as autophosphorylated tyrosine kinase receptors (6). This association leads to the stimulation of the kinase activities of the p110 subunit (6, 7). Since PI 3-kinase is activated by a large range of peptide growth factors, this enzyme activity appears to be implicated in various cellular responses including promotion of cell growth, regulation of cell differentiation, and metabolism (for review see Ref. 6). Despite this, each growth factor triggers distinct and specific biological responses in each particular cell type. In the present study, we looked at the effects of a prior stimulation by platelet-derived growth factor (PDGF) on the further ability of insulin to activate PI 3-kinase. We took advantage of the 3T3-L1 adipocytes where PI 3-kinase can be activated by both insulin and PDGF but not by EGF (8, 9). When PDGF stimulates tyrosine phosphorylation of its receptor, the p85 regulatory subunit of PI 3-kinase associates to phosphorylated Tyr-Xaa-Xaa-Met motifs of the PDGF recepto...

Insulin-like Growth Factor-binding Protein-3 Activates a Phosphotyrosine Phosphatase

“…Preincubation of MCF-7 cells with IGFBP-3 was found to inhibit IGF-IR activation independently of IGFBP-3's ability to bind IGFs (13) and possibly involving conformational changes (14). IGFBP-3 could also alter IRS-1 phosphorylation, as shown in other systems where serine/threonine phosphorylation inhibits tyrosine phosphorylation in response to insulin, hence down-regulating subsequent insulin signaling (21,22). However, this appeared unlikely, since serine/threonine phosphorylation of IRS-1 results in an increase of its molecular mass, which is visible in SDS-PAGE, but which did not occur in our experiments.…”

Transactivation of ErbB2 and ErbB3 by Tumor Necrosis Factor-α and Anisomycin Leads to Impaired Insulin Signaling through Serine/Threonine Phosphorylation of IRS Proteins

“…This feature is also shared by several growth factors that inhibit insulin signaling through serine phosphorylation of IRS-1. Platelet-derived growth factor inhibits insulin-induced tyrosine phosphorylation of IRS-1 in 3T3-L1 adipocytes, and PI3K inhibitors abrogate this effect (42)(43)(44). In the same cells EGF reduces tyrosine phosphorylation of IRS-1 via an MEK-ERK1/ 2-dependent pathway (25).…”

A Monoclonal Antibody to Visualize PtdIns(3,4,5)P3in Cells

“…Previous studies showed that insulin stimulation resulted in a slow rise and sustained PI 3-kinase activity in 3T3-L1 cells, whereas PDGF resulted in rapid and transient PI 3-kinase activity (Ricort et al 1997). Visualization of PtdIns(3,4,5)P 3 at the leading edge of motile cells can be accomplished with either a GFP-PH domain (Rickert et al 2000) or with the RC6F8 IgM described herein (H. Bourne, personal communication).…”